Crystallization of the 43 kDa ATPase domain of Thermus thermophilus gyrase B in complex with novobiocin.

The 43 kDa ATPase domain of Thermus thermophilus gyrase B was overproduced in Escherichia coli and a three-step purification protocol yielded large quantities of highly purified enzyme which remained stable for weeks. Crystals of the 43 kDa domain in complex with novobiocin, one of the most potent inhibitors of bacterial topoisomerases, were obtained. Crystals obtained in the presence of PEG 8000 do not diffract, but a different crystal form was obtained using sodium formate as a precipitating agent. The plate-shaped crystals, which were less than 10 microm in thickness, could be cryocooled directly from the mother liquor and a full diffraction data set was collected to 2.3 A allowing the determination of the first structure of a gyrase B 43K domain in complex with a coumarin.

ICBP90, a novel human CCAAT binding protein, involved in the regulation of topoisomerase IIalpha expression.

The one-hybrid system with an inverted CCAAT box as the DNA target sequence was used to identify proteins acting on key DNA sequences of the promoter of the topoisomerase IIalpha gene. Screening of cDNA libraries from the leukemia Jurkat cell line and from the adult human thymus resulted in the isolation of a novel protein of 793 amino acids (89,758 Da). This protein has in vitro CCAAT binding properties and has been called ICBP90. Adult thymus, fetal thymus, fetal liver, and bone marrow, known as active tissues in terms of cell proliferation, are the tissues richest in ICBP90 mRNA. In contrast, highly differentiated tissues and cells such as the central nervous system and peripheral leukocytes are free of ICBP90 mRNA. Western blotting experiments showed a simultaneous expression of topoisomerase IIalpha and ICBP90 in proliferating human lung fibroblasts. Simultaneous expression of both proteins has also been observed in HeLa cells, but in both proliferating and confluent cells. Overexpression of ICBP90 in COS-1-transfected cells induced an enhanced expression of endogenous topoisomerase IIalpha. Immunohistochemistry experiments showed that topoisomerase IIalpha and ICBP90 were coexpressed in proliferating areas of paraffin-embedded human appendix tissues and in high-grade breast carcinoma tissues. We have identified ICBP90, which is a novel CCAAT binding protein, and our results suggest that it may be involved in topoisomerase IIalpha expression. ICBP90 may also be useful as a new proliferation marker for cancer tissues.

Cloning of Schizosaccharomyces pombe bio2 by heterologous complementation of a Saccharomyces cerevisiae mutant.

A Saccharomyces cerevisiae mutant affected in the last step of the biotin biosynthesis pathway was isolated by using a transposon mutagenesis method. The gene BIO2, encoding a biotin synthase, is shown to be interrupted in this mutant. Heterologous complementation experiment allowed the cloning and the characterization of a novel bio gene: bio2, encoding biotin synthase from Schizosaccharomyces pombe.

Routine detection of Salmonella species in water: comparative evaluation of the ISO and PROBELIA polymerase chain reaction methods.

Water is one of the main transmission routes for Salmonella spp., the causative agents of salmonellosis in humans and animals. This worldwide sanitary problem requires rapid and accurate analyses to be realized so that advisories for exposed people are timely and reliable. Because the traditional method, the ISO 6340 method, does not meet the criterion of rapidity, there was a need for a quicker method. To fulfill this need, a comparative evaluation between the ISO method and one based on polymerase chain reaction (PCR), the PROBELIA, was performed. Waters from different origins were tested either directly or after artificial contamination with selected Salmonella strains isolated from the environment. The results clearly demonstrate that the PCR-based method can advantageously replace the ISO 6340 method. It is quicker, less labor intensive, reproducible, and provides results that match perfectly to those obtained by the ISO method. Furthermore, the PROBELIA method meets the requirements for an efficient sanitary survey: high numbers of samples processed at the same time, reduced cost, and results within 2 days.

Characterization of the biotin biosynthesis pathway in Saccharomyces cerevisiae and evidence for a cluster containing BIO5, a novel gene involved in vitamer uptake.

An engineered mutant of Saccharomyces cerevisiae affected in biotin biosynthesis has been isolated. This mutant allowed the characterization of a bio cluster (BIO3-4-5). We demonstrate that BIO3 (YNR058w) and BIO4 (YNR057c) encode, respectively, a 7, 8-diaminopelargonic acid aminotransferase and a dethiobiotin synthase, involved in the biotin biosynthesis pathway. A novel gene, BIO5 (YNR056c), is present immediately downstream from BIO4. This gene encodes Bio5p, a protein with 11 putative transmembrane regions. Uptake experiments performed with labeled 7-keto 8-aminopelargonic acid indicate that Bio5p is responsible for transport into the cell of 7-keto 8-aminopelargonic acid.

Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway.

Cinnamate 4-hydroxylase [CA4H; trans-cinnamate,NADPH:oxygen oxidoreductase (4-hydroxylating), EC 1.14.13.11] is a cytochrome P450 that catalyzes the first oxygenation step of the general phenylpropanoid metabolism in higher plants. The compounds formed are essential for lignification and defense against predators and pathogens. We recently reported the purification of this enzyme from Mn(2+)-induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. Highly selective polyclonal antibodies raised against the purified protein were used to screen a lambda gt11 cDNA expression library from wound-induced Jerusalem artichoke, allowing isolation of a 1130-base-pair insert. Typical P450 domains were identified in this incomplete sequence, which was used as a probe for the isolation of a 1.7-kilobase clone in a lambda gt10 library. A full-length open reading frame of 1515 base pairs, encoding a P450 protein of 505 residues (M(r) = 57,927), was sequenced. The N terminus, essentially composed of hydrophobic residues, matches perfectly the microsequenced N terminus of the purified protein. The calculated pI is 9.78, in agreement with the chromatographic behavior and two-dimensional electrophoretic analysis of CA4H. Synthesis of the corresponding mRNA is induced in wounded plant tissues, in correlation with CA4H enzymatic activity. This P450 protein exhibits the most similarity (28% amino acid identity) with avocado CYP71, but also good similarity with CYP17 and CYP21, or with CYP1 and CYP2 families. According to current criteria, it qualifies as a member of a new P450 family.

In situ mapping of the chicken progesterone receptor gene and the ovalbumin gene.

The chicken progesterone receptor (cPR) gene and the ovalbumin (OA) gene, a target of cPR regulation, have been mapped via fluorescent in situ hybridization to the two largest chromosomes of the chicken karyotype. cPR is subtelomeric on the long arm of chromosome 1 and OA is on the long arm of chromosome 2, close to the centromere. A 35-kb cosmid probe for the cPR gene and two genomic fragments of 9.2 and 15 kb for the OA gene were biotin-labeled for nonradioactive localization of the two chicken loci.

Microchromosomal assignment of the chicken ovotransferrin and adenylate kinase genes.

Ovotransferrin, an egg-white protein implicated in the transfer of trace elements from the hen oviduct to the developing avian embryo, and cytosolic adenylate kinase, an essential enzyme involved in the interconversion of adenine nucleotides in energetically active tissues, have been mapped to two separate chicken microchromosomes by fluorescent in situ hybridization. Considering present and previous data, the possibility of loss of intron material resulting in the compactation of genes in chicken microchromosomes is briefly discussed.

Chicken poly(ADP-ribose) synthetase: complete deduced amino acid sequence and comparison with mammalian enzyme sequences.

The complete nucleotide (nt) sequence of the cDNA encoding the chicken poly(ADP-ribose) synthetase has been determined. Positive clones overlapping the 5' region or the 3' region of the cDNA have been isolated from a lambda gt 10 hen oviduct cDNA library using two human cDNA probes. The missing middle portion has been obtained by the polymerase chain reaction procedure. A single 3033-nt open reading frame from start codon to stop codon encodes a sequence of 1011 amino acid residues. The alignment of this sequence with those from human and mouse reveals overall identities of 79% and 77%, respectively. However, an identity of about 82% is obtained in the DNA-binding domain within the two zinc fingers, and an even higher similarity (85-87%) is observed in the NAD-binding domain. The isolated clones consistently hybridize on chicken Northern blots to an mRNA species of about 4 kb, whereas they do not cross-hybridize with RNA blots of Drosophila melanogaster. Thus, it appears that, even if the functional properties of the enzyme are maintained, the cDNA identity will be much decreased in nonvertebrate organisms.

Isolation and characterization of an expressed hypervariable gene coding for a breast-cancer-associated antigen.

A human gene and cDNA coding for a breast-cancer-associated antigen (H23Ag) were isolated and characterized. The gene contains two exons and one intron. Part of the second exon is a tandem repeat array (TRA) consisting of multiple 60-bp G + C-rich units. We report here the characterization of unique sequences that are found in the H23Ag gene and cDNA, in addition to the 60-bp repeats. Analysis of the cDNA sequences revealed a putative ATG start codon preceded by two overlapping initiation consensus sequences (CCACC). The open reading frame determines an amino acid (aa) sequence consisting of three regions. The first region contains an initiating methionine and a highly hydrophobic putative signal peptide. This is followed by a variable number of highly conserved 20-aa repeat units (TRA). The last region, C-terminal to TRA, contains four potential N-linked glycosylation sites. The genomic nucleotide sequences demonstrate a putative promoter region that includes a 'TATA' box. A putative estrogen regulatory element is located 5' to the promoter region. The characterization of the gene and cDNA coding for the H23Ag presented here, may help to elucidate its possible function in human breast cancer.

Characterization of multiple mRNAs originating from the chicken progesterone receptor gene. Evidence for a specific transcript encoding form A.

The structure of the 42-kilobase (kb) long chicken progesterone receptor (cPR) gene and of all six transcripts that are detectable on Northern blots was determined. The first of 8 exons encodes the N-terminal region A/B which is highly divergent among different species and contains a constitutive transcription activation function. The DNA (DBD)- and hormone-binding domains (HBD) are assembled from 2 and 5 exons, respectively, with the individual "zinc fingers" of the DBD encoded by separate exons. In addition to the previously described 4.5-kb cPR mRNA species, alternative polyadenylation, splicing variation, and "5'-truncation" lead to the generation of 5 further mRNAs. Most importantly, this 5'-truncation produces, by an as yet unidentified mechanism, an abundant transcript which encodes form A but not form B of cPR. Lack of splicing at the exon 2 splice-donor and polyadenylation due to a signal site in the second intron generates a previously undetected 3.4-kb mRNA species. The corresponding cDNA was sequenced in its entirety and shown to encode only region A/B and the N-terminal "finger" of the DBD. Alternative polyadenylation upstream of the signal site for the 4.5-kb mRNA is responsible for the appearance of a 3.3-kb mRNA. The longest cPR mRNA (8.2 kb) originates from a transcription termination point more than 3 kb downstream of the 4.5-kb mRNA 3'-end. Finally, the primary sequence of more than 2 kb upstream sequences of the cPR gene, containing several consensus hexamer progestin/glucocorticoid receptor-binding sites (PRE/GRE and putative Sp1 binding motifs, is discussed.

Sequence of human villin: a large duplicated domain homologous with other actin-severing proteins and a unique small carboxy-terminal domain related to villin specificity.

Villin is a calcium-regulated actin-binding protein that caps, severs, and bundles actin filaments in vitro. This 92,500-D protein is a major constituent of the actin bundles within the microvilli of the brush border surface of intestinal and kidney proximal tubule cells. Villin is a very early marker of cells involved in absorption and its expression is highly increased during intestinal cell differentiation. The amino acid sequence deduced from the cDNA sequence revealed that human villin is composed of three domains. The first two domains appear as the result of a duplication: their structural organization is similar. We can then define a basic unit in which a slightly hydrophilic motif is followed by three hydrophobic motifs, similar between themselves and regularly spaced. The duplicated domain is highly homologous to three other actin-severing proteins and this basic structure represents the whole molecule in severin and fragmin, while two basic units compose gelsolin. The third domain which is carboxy terminal is villin specific: it is unique among actin modulating proteins so far known. It could account for its actin-binding properties (dual regulation by calcium of severing and bundling activities). We propose that it may also be related to the subcellular localization of villin in different epithelial cell types.

The 5' flanking region of the human pS2 gene mediates its transcriptional activation by estrogen in MCF-7 cells.

The pS2 gene is transcriptionally induced by oestrogen in the human breast cancer cell line MCF-7. We demonstrate here that the 5' flanking sequences (-3000 to +10 bp) of the pS2 gene possess the properties of an oestrogen-inducible promoter. Interestingly, this oestrogen induction could not be demonstrated in transient transfection assays in MCF-7 cells, but only in stably transformed MCF-7 cells, which suggests that some factors responsible for oestrogen induction may be present in limiting amounts in these cells and absent in HeLa cells.

The chicken progesterone receptor: sequence, expression and functional analysis.

The complete mRNA sequence of the chicken progesterone receptor (cPR) has been determined. Expression of the cloned cDNA both in vivo and in vitro produces a protein that has the same apparent mol. wt on SDS--polyacrylamide gels as the 'natural' cPR form B (109 kd) as determined by immunoblotting and photoaffinity labelling. When expressed in HeLa or in Cos-1 cells the 'cloned' cPR displays hormone binding characteristics indistinguishable from the 'natural' receptor and, in the presence of progestins, exhibits 'tight nuclear binding'. A protein corresponding in size to the cPR form A (79 kd) could be detected by expressing in vivo and in vitro an N-terminally truncated cPR starting at methionine 128. A protein of the same apparent mol. wt results from internal initiation during in vitro translation. In contrast, such a protein was barely detectable after in vivo expression of the cPR cDNA in Cos-1 cells. These results suggest that form A is generated by an oviduct cell specific process involving either internal initiation of translation and/or proteolysis in the vicinity of methionine-128. The cPR contains two highly conserved regions C and E, a characteristic of the steroid/thyroid hormone receptor supergene family. By expression of a series of cPR deletion mutants, region E could be defined as the hormone binding domain whereas region C is indispensable for the tight nuclear association of the progestin-receptor complex. In the presence of progestins, the cloned cPR efficiently trans-activates transcription from the long terminal repeat region (LTR) of the mouse mammary tumor virus (MMTV). Deletion of the entire N-terminal region A/B or of the hormone binding domain E results in a 100-fold reduction of transcriptional activation. No stimulation of transcription can be detected when the C-terminal deletion extends into region C, indicating that this region is involved in the recognition of the hormone responsive element (HRE) of the MMTV LTR.

Cloning of the saliva-interacting protein gene from Streptococcus mutans.

Genomic libraries from Streptococcus mutans OMZ175 were constructed in bacteriophage vectors. DNA fragments 1 to 2 kilobases in length were cloned in expression vector lambda gt11. S. mutans DNA fragments 15 to 20 kilobases in length were inserted in the BamHI site of phage EMBL3. Rabbit antiserum raised against an S. mutans saliva-interacting protein with a molecular weight of 74,000, designated 74K SR, was used to screen the lambda gt11 library. A recombinant phage carrying an S. mutans DNA sequence of 1.45 kilobases, lambda SmAD2, was detected and isolated. This fragment, named SmAD2, was used to construct the recombinant expression plasmid pSAD2-4 which encoded for the expression of a 60,000-molecular-weight protein controlled by the beta-galactosidase promoter from plasmid pUC8. The SmAD2 fragment and polyclonal anti-74K SR antibodies were used to screen the EMBL3 library. A total coincidence between the screening with antibodies and the DNA probe was observed, and two phages, lambda SmAD9 and lambda SmAD10, were isolated. They contained a common S. mutans DNA sequence of about 11.8 kilobases and coded for a protein with a molecular weight of about 195,000, which comigrated with a protein of an S. mutans cell wall extract. The expressed protein was purified, and a very strong relationship with the S. mutans 74K SR protein was found by competitive enzyme-linked immunosorbent assay. Thus, cloning of the 74K SR gene allowed us to demonstrate that the saliva receptor appears to be a part of an S. mutans precursor molecule with a molecular mass of 195,000 daltons.

Isolation of high-molecular-weight DNA from eukaryotic cells by formamide treatment and dialysis.

A new method for the isolation of high-molecular-weight DNA from eukaryotic cells is presented. It is based on formamide treatment and extensive dialysis of cellular proteinase K digests. This procedure consists mainly of a series of incubations allowing simultaneous preparation of several DNAs which can then be restricted, ligated, hybridized, and cloned.

Structure of the human oestrogen-responsive gene pS2.

The human pS2 gene, whose expression is restricted to breast cancer cells, and whose transcription is induced by oestrogen in the human breast cancer cell line MCF-7, has been cloned from both placental and MCF-7 cell DNA. The exon-intron organization has been established by electron microscopy using genomic DNA-cDNA or -mRNA hybrid duplexes and by sequencing the exons and exon-intron junctions. The overall organization within and around the pS2 gene is the same in placental and MCF-7 cell DNA and the exonic sequences are identical to those previously determined from the cDNA. The 5'-flanking region of the pS2 gene is also identical (with the exception of two base transitions) in the two tissues. Thus no gene rearrangement nor sequence modification has occurred in the pS2 gene of the malignant and polyploid MCF-7 cells. A TATA-box, a CAAT-box and a GC-rich motif are present in the 5'-flanking region of the pS2 gene, but the latter motif is unusually located between the TATA-box and the capsite. No significant homology could be detected between the 5' flanking sequences of the pS2 gene and those of other oestrogen-responsive genes from different species.